PROJECT SUMMARY Limb loss affects nearly 2 million Americans and comes with complications such as infection, pain, and blood clots, in addition to serious psychological impact. Currently, the standard of care following limb loss is the use of a prosthetic. The ideal response following limb loss would be the regeneration of an exact replicate of the lost limb. This is not the response humans make, but other species such salamanders have the ability to perfectly regenerate lost appendages. Therefore, understanding the remarkable ability of salamanders to regenerate lost limbs may elucidate mechanisms to unlock the regenerative capacity of humans. Following limb amputation salamanders form a structure called the blastema. The blastema is a pool of tissue specific progenitor cells that are responsible for regenerating the lost limb. Recent gains in molecular biology has allowed for discovery of genes that are enriched and specific to the regenerating limb. We have recently identified von Willebrand Factor D and EGF domains (VWDE) as a highly blastema enriched and specific gene. We hypothesize that the blastema contains a pool of transcriptionally distinct progenitor cells and pan- blastemal genes, such as VWDE, support this niche. Our first aim will evaluate the role and functional domains of VWDE in blastema cell function and mammalian cell differentiation. Our second aim will work to uncover other progenitor cells (i.e. dermis, cartilage, Schwann cells) using single cell RNA-sequencing to elucidate the cellular heterogeneity of the blastema. These studies will provide essential information on a novel gene and work to uncover different progenitor cells required for limb regeneration in salamanders. These gains in understanding of salamander limb regeneration will put us closer to unlocking the ability to promote regeneration in humans.